The goal of this research is to define and characterize the neural networks governing two sequentially-linked behavioral programs in the fly. The first is an adaptive, environmentally-sensitive program that mediates the search for safe surroundings, and the second is a hormonally-driven program that serves to expand the recently developed wings of the newly emerged adult. The first program is used to find a safe perch from which the immobile fly can expand its wings, and the second program initiates expansion. Because wing expansion must be undertaken within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. Our laboratory's approach to understanding how the Drosophila nervous system produces behavioral sequences crucially depends on genetic techniques that allow specific subsets of brain cells to be turned off or on in freely behaving animals. This approach requires genetic tools for both suppressing and stimulating brain cell activity, as well as tools to target these manipulations to the desired subset of cells. Making such tools, both for our circuit-mapping activities and also for those of other laboratories, is another principal goal of our research. Much of our own recent research has been driven by the development of a technique for acute neuronal activation using the mammalian cold-sensitive channel TRPM8, which we introduced in 2009 (Peabody et al., J Neurosci., 29:3343-53). Last year we used this technique to identify a pair of command neurons for wing expansion behavior, which are located in the subesophageal ganglion of the fly nervous system (Luan et al., J Neurosci., 2012, 32:880-9). More recently we have used TRPM8 to determine when these command neurons become competent to drive the wing expansion program during development. To do so, we have stimulated the command neurons at developmentally ectopic times and monitored the neuroendocrine and behavioral outputs of the wing expansion network. We found that the network is suppressed--likely downstream of the command neurons--prior to emergence of the fruit fly from its pupal case, and that the act of emergence, or some process closely associated with it, lifts this suppression. Our results thus provide insight into how nervous systems use inhibitory mechanisms to assemble motor programs into correctly ordered behavioral sequences. This work is the subject of a manuscript currently under revision at the Journal of Experimental Biology. The ability to acutely activate neurons using TRPM8 also featured centrally in the research described in two other manuscripts accepted for publication this year. One was published in the journal Nature (Flood et al., Nature, 2013 499:83-7), and the other is currently in press at the journal G3. Both of these papers are the result of a productive collaboration with the laboratory of Dr. Moto Yoshihara at the University of Massachusetts Medical School. Dr. Yoshihara's laboratory, like mine, has an interest in identifying core circuit components responsible for generating simple behaviors in the fly. Our collaborative effort to identify command neurons for such behaviors is described in the G3 paper. The paper published in Nature highlights one of the principal successes of this approach and provides a detailed characterization of a pair of command neurons that controls feeding behavior in Drosophila. Much of the rest of the laboratory's work during the last year has capitalized on a second technical advance, which we reported in 2012 in the journal Genetics (Diao & White, Genetics, 2012, 190:1139-44). This so-called T2A-GIFF technique is a novel strategy for coupling the expression of the transcription factor Gal4 to the expression of endogenous genes of interest using the self-cleaving capacity of the viral T2A peptide. We first used this strategy to make a fly line that co-expresses Gal4 in cells that express the receptor for bursicon, the hormone that governs wing expansion. In addition to using this line to characterize the functional roles of bursicon receptor-expressing neurons in the wing expansion circuit (work which we are currently preparing for publication), we have also identified a novel set of cells in the fly gut that use bursicon signaling to regulate stem cell turnover. This work has been carried out in collaboration with the laboratory of Dr. Marcos Vidal at the Beatson Institute in Scotland and is the subject of a manuscript currently under review at the journal Science. Another manuscript, in which the T2A technique is exploited to characterize the circuitry underlying color processing in the Drosophila brain, is currently in revision at the journal Neuron. The latter work is the result of a collaboration with the laboratory of Dr. Chi-hon Lee at NICHD. In addition to exploiting the T2A-GIFF technique to investigate specific biological questions, we have also spent considerable effort over the last year further developing this technique. To extend its range of application, we have developed a broad toolkit of plasmids and fly lines that permits the T2A-GIFF technology to be easily used in conjunction with the MiMIC transposon-containing lines produced by the Drosophila Gene Disruption Project. Our MiMIC-T2A-mediated In-Frame Fusion method (i.e. MiMIC-TIFF) allows Gal4 and numerous other transgenes to be expressed in the same pattern as endogenous genes that contain MiMIC insertions. The method works for any MiMIC insertion that lies within the coding intron of a gene of interest and uses recombinase-mediated cassette exchange to replace the MiMIC insert with a T2A fusion construct preceded by a splice acceptor site. Currently, there are well over 1000 suitable MiMIC insertions available for use with this technique. We have tested the technique with T2A constructs that permit expression of not only Gal4, but also Gal80 and components of the Split Gal4 system, which we introduced in 2006 (Luan et al., 2006, Neuron, 52:425-36). In all cases, we observe high fidelity expression of the T2A constructs in the same patterns as the endogenous genes into which they have been inserted. A manuscript describing this technique is currently in preparation and is expected to be submitted later this year. Given the ever-increasing number of MiMIC lines available, the MiMIC-TIFF method should be of very broad use to the Drosophila research community. In summary, we have made substantial progress during the last year in elucidating, or helping to elucidate, several behavioral circuits in the Drosophila nervous system, including the one that supports wing expansion. At the same time, we have continued to develop tools that will support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the wing expansion circuit, our work should provide insight into the principles used by all nervous systems to generate and organize behavior. In addition, our work should serve as a proof of concept of a circuit-mapping approach that can be extended to studies of mammalian behavior using similar tools that are becoming available for vertebrate organisms.